金针菇基因组测序与组装策略分析

采用二代和三代测序技术分别对金针菇单核体菌株“6-3”进行测序,应用4种组装策略进行基因组的de novo组装,对比组装效果。基因组组装的参数方面,仅使用二代测序组装的效果最差,长度大于10kb的Contig全长只有24.6Mb,Contig N50只有23kb,组装率只有59.27%。采用三代组装二代校正的组装策略效果最好,长度大于10kb的Contig全长为38.3Mb,Contig N50为2.8Mb,组装率高达92.16%。保守单拷贝基因拼接效果方面,4种组装策略获得基因组序列与BUSCO数据库里的担子菌的保守单拷贝基因比对,基因完整性均大于94%。在组装准确性方面,经过PCR扩增、Sanger测序验证,三代组装二代校正的基因组序列完整并且连续,同时序列上碱基的SNP、InDel数量最少。综上所述,三代组装二代校正得到的基因组序列具有Contig N50值大、组装率高、碱基准确性高的特点,是食用菌基因组测序较为理想的方案。

Genome sequencing and assembly strategy analyses of Flammulina filiformis

Next-generation sequencing (NGS) and third-generation sequencing (TGS) technology were used to sequence Flammulina filiformis monokaryon strain 6-3, and de novo assembly using four assembly strategies was performed, and then the quality of assembly was evaluated. As regards parameter of assembly, the quality of NGS assembly is the worst. The total length of contig longer than 10kb is only 24.6Mb; contig N50 is only 23kb, and only 59.27% of data is assembled into contigs. The quality of TGS assembly rectified with NGS is the best. The total length of the contigs longer than 10kb is 38.3Mb; contig N50 is 2.8Mb, and up to 92.16% of data is assembled into contigs. Regarding quality of conserved single-copy gene assembly, the assembled sequences obtained from all four strategies were aligned with conserved single-copy gene of basidiomycetes in the BUSCO database. It is showed that gene completeness for each strategy is over 94%. Verification by PCR and Sanger sequencing indicated that TGS assembly rectified with NGS yielded complete and continuous genome sequence with the least of SNP and InDel. TGS assembly rectified with NGS manifests high N50, high genome assembly ratio, and high base accuracy, and it is a relatively ideal scheme for edible fungal genome sequencing.